Scanning spectrophotometry and spectrophotometric
determination of concentration
BCH 333 [practical]
:
Objectives
1. What is absorption spectrum and determination of λmax.
2. standard curve and determination of concentration of an unknown solution.
How a spectrophotometer works
The spectrophotometer: it can be used to measure the amount of light absorbed
by a solution. How?
-Using the spectrophotometer, we can quantitatively measure absorbance, and this
information can be used to determine the concentration of the absorbing molecule.
-A more concentrated solution will absorb more light and transmits less .[why?]
So, the more concentrated solution  high absorbance value.
And Less concentrated solution  less absorbance value.
Absorption spectrum:
The curve that display the action or behavior of absorption of molecule[solute] at
different weave lengths. Every molecule has its own Absorption spectrum, So it’s act as
fingerprint for each molecule.
λmax: it is the weave length at which the molecule has the maximum absorbance at this
weave length.[best absorbance]
[the wavelength of maximum absorption (λmax)]
Absorption spectrum curve and determination of λmax.
What is the benefit of studying the Absorption spectrum :
-λmax.
-used to identify substances.
-It also can be used to know if there is any contamination with another molecule.
How does a spectrophotometer work?
http://www.youtube.com/watch?v=pxC6F7bK8CU
Beer-Lambert law:
Principle:
The absorption of light by a solution is described by the Beer-Lambert law:
The law implies that there is linear relationship between absorbance and
concentration of an absorbing species.
A = εlc
A= is the absorbance.
ε = extinction(absorption) coefficient.
l = length of the light path through the solution.
c = concentration of the absorbing substance.
So, what does standard curve mean?
A standard curve:
- It is a graph that shows the relationship between different known concentrations of a
substance and the absorbance at a specific wave length.
-Standard curve are most commonly used to determine the concentration of a substance,
using serial dilution of solutions of known concentrations.
-So, what is the principle of a standard curve???
So, what is standard solutions???
standard solution:
is a solution containing a precisely known concentration of an element or a substance.
How to determine unknown concentration,
of a solution with known absorbance value?
If the unknown solution has absorbance value =0.45,
the conc. From the curve will be ......
BSA standard curve
0.9
0.8
0.7
Absorbance of the
” unknown solution”
O.D at....nm
0.6
0.5
0.4
0.3
Concentration of the
” unknown solution”
0.2
0.1
0
0
1
2
3
4
5
6
BSA [mg/ml]
[known concentration of BSA]
7
8
9
To determine the concentration of an unknown solution:
From standard curve:
-Measure the absorbance of the
“unknown solution” in order to
determine the concentration.
-…
Beer-Lambert law:
Using available information of any
standard solution to determine the “ε”,
then using these information to get the
unknown concentration. Using:A = εlc
Note: “ε” will changed when the weave
length changed.
A-Absorption spectrum of bromophenol blue:
Draw absorption spectrum curve[absorbance against wavelength] of bromophenol blue then
determine λmax.
Wave length [n.m]
Absorbance
320
0.105
340
0.110
360
0.120
380
0.167
400
0.251
420
0.334
440
0.357
460
0.306
480
0.203
500
0.113
520
0.056
540
0.037
560
0.038
580
0.051
600
0.055
620
0.019
λmax
λmax for bromophenol blue =440nm
2. Standard curve
a) Set up 8 test tubes as follows:
Tube No.
7.5x10 - 5M
bromophenol blue (ml)
[std.]
95% ethanol
(ml)
0.1 M citrate buffer
[pH 2.4] (ml)
Sample
With unknown
concentration
(ml)
1
2
3
4
5
6
7
[unknown]
0.1
0.9
9.0
-
0.2
0.8
9.0
-
0.4
0.6
9.0
-
0.6
0.4
9.0
-
0.8
0.2
9.0
-
1.0
-
9.0
-
-
-
9.0
1.0
b) Mix and measure the absorbance of all the tubes at 430 nm against a water blank.
Results:
Tube No.
Molar concentration of
bromophenol blue x 10 - 6
Absorbance at 430 nm
1
0.75
0.171
2
1.5
0.391
3
3
0.806
4
4.5
1.243
5
6
1.639
6
7.5
1.996
7[unknown]
...................
1.505
b-Plot a standard curve of absorbance against Molar concentration of bromophenol
blue x10 – 6.
c-From the curve determine the molar concentration of the unknown sample (tube 7).
- Molar concentration of bromophenol blue in unknown sample (tube 7).=……… x 10 – 6
Standard curve animation:
http://www.wellesley.edu/Biology/Concepts/Html/standardcurve.html
Spectrophotometry Introduction:
http://www.youtube.com/watch?v=qbCZbP6_j48
Spectrophotometry Example
http://www.youtube.com/watch?v=VqAa_cmZ7OY&feature=relmfu